Proc. Nati. Acad. Sci. USA Vol. 90, pp. 883-887, February 1993 Biochemistry Pattern of aromatic and hydrophobic amino acids critical for one of two subdomains of the VP16 transcriptional activator (transcriptional activation/herpes simplex virus/site-directed mutagenesis/virion protein Vmw65/a-trans-inducing factor) JEFFREY L. REGIER*, FAN SHENt, AND STEVEN J. TRIEZENBERG*t* *Genetics Program and tDepartment of Biochemistry, Michigan State University, East Lansing, MI 48824-1319 Communicated by Steven McKnight, September 29, 1992 (receivedfor review July 14, 1992) ABSTRACT Structural features of the transcriptional ac- tivation domain ofthe herpes simplex virion protein VP16 were I examined by oligonucleotide-directed mutagenesis. Extensive 413 456 490 mutagenesis at position 442 of the truncated VP16 activation Leu Asp Asp Phe Asp LeuAspMet MtAla Asp Phe Glu Phe Glu Gln Met domain (A456), normally occupied by a phenylalanine residue, 439 442 444 473 475 demonstrated the importance ofan aromatic amino acid at that position. On the basis of an alignment of the VP16 sequence FIG. 1. Schematic representation of the VP16 activation domain surrounding Phe-442 and the sequences of other transcrip- (amino acids 413-490). The truncated VP16 activation domain (A456) tional activation domains, we subjected leucine residues at lacks residues 457-490 (24, 31). Portions ofthe amino acid sequence positions 439 and 444 of VP16 to mutagenesis. Results from are shown, using hollow type for hydrophobic amino acids and bold these experiments suggest that bulky hydrophobic residues type for acidic amino acids. flanking Phe-442 also contribute signifucantly to the function of In the case of VP16, the amino-terminal region of the protein the truncated VP16 activation domain. Restoration of amino interacts with host cell factors that bind to IE gene promoter acids 457-490 to various Phe-442 mutants partially restored elements (18-23), while the transcription activation function activity. Although the pattern of amino acids surrounding resides in the carboxyl-terminal 80 amino acids (24-28). Phe-473 resembles that surrounding Phe-442, mutations of Various transcriptional activation domains, including the Phe-473 did not dramatically affect activity; in fact, Phe-475 domain of are rich appears more sensitive to mutations than does Phe-473. We VP16, in acidic amino acids (29, 30). infer that the two regions of VP16 (amino acids 413-456 and Previous work from our laboratory (31) demonstrated that net 457-490) possess unique structural features, although neither negative charge was necessary but not sufficient to account is likely to be an amphipathic a-helix or an "acidic blob." for the strong activation ability of VP16; other elements of These results, considered with previous in vitro activation and protein structure also contributed to the activity. Giniger and inhibition studies, suggest that the two subdomains of VP16 Ptashne (32) proposed that acidic activation domains form affect transcription by different mechanis. amphipathic a-helices, and indeed insertion of a potentially helix-breaking proline residue at position 442 ofthe truncated VP16 The mechanisms that regulate the rate of transcriptional activation domain (codons 413-456) abolished activity. initiation depend in part on factors termed transcriptional However, replacing this proline residue with helix- activators (1, 2). Several models have been proposed to compatible but nonaromatic amino acid residues (Ala, Ser) explain how activators work. They may function by directly did not restore activity to the VP16 activation domain. or indirectly contacting a component of the basal transcrip- Furthermore, substitution of two prolines simultaneously at tion complex, thereby speeding up the ordered assembly of positions 432 and 436 within the predicted a-helix also had no that complex at the promoter (1-6). Activators could also effect on activity. Here we report further mutations of VP16 increase the rate of transcriptional initiation by relieving the that demonstrate the importance of an aromatic moiety at inhibition caused by histones, thus allowing the transcription position 442 and strengthen the suggestion that the activity of complex to form (7-11). A third model posits that activators this domain does not depend upon an amphipathic a-helix. transform a preinitiation or initiation complex into an elon- After the discovery of the importance of Phe-442, the gation complex, allowing transcription to proceed (12-14). amino acid sequence of the VP16 activation domain was These models need not be mutually exclusive; in fact, evi- compared to the sequences ofother transcriptional activation dence exists for the participation of the transcriptional acti- domains. The sequences of several transcriptional activation vation domain of VP16 (a prototypical activator) in several of domains were aligned, using as a guide six bulky hydrophobic these mechanisms. Because an understanding of how acti- residues of the VP16 activation domain (31). In a number of vators function is crucial to our understanding of transcrip- different types of activation domains, bulky hydrophobic tional regulation, we have undertaken a detailed and system- residues are observed at positions that can be aligned with atic mutagenesis study of VP16. Leu-439 and Leu-444 of VP16, on either side of the critical VP16 [also known as a-trans-inducing factor (a-TIF) and Phe-442 (see Fig. 1). The only direct test of this pattern in Vmw65] is the component ofthe herpes simplex virus 1 virion another activation domain has recently been reported by that specifically activates transcription ofthe viral immediate Hardwick et al. (33), who found that this pattern is important early (IE) genes (15, 16). Many transcriptional activator for activation by the Rta protein ofEpstein-Barr virus. In the proteins have two domains: one conferring specific associa- work reported herein, these two leucine residues of VP16 tion with promoter sequences, usually a DNA-binding do- were also targeted for mutagenesis; substitution of small main, and a second domain for regulatory function (1, 2, 17). hydrophobic or hydrophilic amino acids for either leucine greatly decreased activity. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: IE, immediate early; tk, thymidine kinase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 883 Downloaded by guest on September 27, 2021 884 Biochemistry: Regier et al. Proc. Natl. Acad. Sci. USA 90 (1993) We also show that the addition of amino acids 457-490 to visualized by using a biotinylated secondary antibody and the truncated VP16 activation domain partially restores the avidin/biotinylated enzyme complex (Vector Laboratories), activities of truncated VP16 mutants inactivated by changes the substrate for which was 4-chloro-1-naphthol. at Phe-442. Within this added region, a phenylalanine at position 473 is found in a context similar to that surrounding Phe-442. Changing this residue to nonaromatic amino acids RESULTS only modestly affected the activity of this region. Instead, a Importance of an Aromatic Amino Acid at Position 442. neighboring phenylalanine (at position 475) was somewhat Previous results from this laboratory (31) suggested that more sensitive to mutations. Our work suggests that two Phe-442 is critical for function of the truncated VP16 activa- regions of the VP16 activation domain have different struc- tion domain (A456). To more thoroughly test this hypothesis, tural elements and may have different functions for transcrip- additional amino acid substitutions were made at position tional activation. 442. Activities ofVP16 mutants were determined by transient transfection assays in which a plasmid expressing the VP16 MATERIALS AND METHODS gene was cotransfected with a reporter plasmid bearing the herpes simplex virus 1 tk gene under the control of a Mutagenesis and Cloning. Sal I/BamHI fragments corre- VP16-responsive promoter and an internal control plasmid sponding to the truncated (codons 411-456) or full-length consisting of the tk gene regulated by the Moloney murine (codons 411-490 plus 7 bp of 3' nontranslated sequence) sarcoma virus long terminal repeat promoter. Total RNA was activation domain of VP16 (24) were inserted into M13mpI9 harvested and the amount of tk RNA was quantitated by (34). Oligonucleotide-directed mutagenesis was performed as primer extension assay and scintillation counting. In our described (31, 35, 36). Mutations were identified by dideoxy sequencing (37), and double-stranded assays, the full-length VP16 protein activates expression of phage DNAs contain- the reporter by 20-fold, and the truncated (A456) protein ing the desired mutant VP16 activation domains were har- vested. The Sal I/BamHI encoding the altered activates approximately 12-fold. fragments Of the 19 possible changes at position 442, 17 have been activation domains were cloned downstream ofcodons 1-410 by using the expression vector pMSVP16 (24). generated and their activities have been tested. Substitution two Transient Transfection Assay. Mouse L cells were grown in of the other aromatic amino acids, tyrosine and tryp- Dulbecco's modified Eagle's medium (GIBCO) supple- tophan, for Phe-442 decreased but did not abolish function (Fig. 2). No other substitution mutants had an mented with 10% fetal calf serum (HyClone). Cells (8 x 105